Studies On The Breeding Of Producing Xylanase Strain And Property Of Xylanase

A preservation strain FL012 which could secrete extracellular xylanase was to be studied. The xylan-degrading bacteria FL012, was identified as Paenibacillus campinasensis based on morphological, biochemical, physiological characteristics and 16S rDNA sequence analysis. To the best of our knowledge, this is the first report of the characteristics of xylanase produced by Paenibacillus campinasensis. After being mutated by the ultraviolet radiation and diethyl sulfate treatment, a mutant strain named D9-38 was obtained, which had a good genetic stability and xylanase production ability. The xylanase activity of mutant strain D9-38 increased to 120.90IU/mL, and increased by 50.09% compared with the original strain FL012.The optimal compositions of the medium for Cl strain producing xylanase are as follows(g/L): wheat bran 7.7, peptone 5.0, yeast extract 5.0, K2HPO4 0.87, MgSO4·7H2O 0.2, CaCO3 12.5(separately autoclaved); The optimal cultivation conditions for D9-38 producing xylanse are the initial pH8.5, fermentation temperature of 37℃, with volume of 35mL/250mL, amount of inoculation of 2.0%(V/V), and shaking cultivation at 150r/min for 50 h. The xylanase activity of fermented broth could reach 266.53IU/mL when D9-38 was incubated in flask under the optimal cultivition conditions.The xylanase was purified to homogeneity by ammonium sulfate fractionation, dialysis and concentrated, and Sephadex G-75 Chromatography, Sephadex G-150 Chromatography. The molecular mass of this xylanase is 21.7 kD. The optimum pH and temperature for the activity are 7.5 and 60℃, respectively. The enzyme is stable over a broad pH range from pH 5.5～9.0 and showes a good thermal stability when incubated at 50℃～60℃. The enzyme activity is strongly inhibited by Hg2+ and enhanced by Mn2+, Ca2+. The Km and Vmax values are 13.87 mg/mL and 47.85μmol/(mL.min) respectively, for oat spelt xylan as the substrate. The xylanase has a good specificity and it has no effect on the carboxymethyl cellulose. The enzymatic hydrolysis products were analyzed by the paper chromatography and by the TLC chromatography. Experimental results demonstrate that this xylanase is an endo-type cleave enzyme.The results show that the mutant D9-38 with a good genetic stability and xylanase production ability, can produce cellulase-free endo-xylanase and it has strong potential for industry application.